Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells

Abstract

To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), d- erythro sphingosine (De SPH), and its three unnatural Stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with ic 50 values of approximately 50 μM in both assays. Treatment of multidrug-resistant MCF-7 ADR cells with SPH Stereoisomers increased vinblastine (VLB) accu- mulation up to 6-fold at 50 μM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7 ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH Stereoisomers. Treatment of MCF-7 cells with SPH Stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [ 3H] VLB to MCF-7 ADR cell membranes or [ 3H]azidopine photoaffinity labeling of P-gp or alter P-gp ATPase activity. These results suggest that SPH isomers are not substrates of P-gp and suggest that modulation of VLB accumulation by SPH Stereoisomers is associated with inhibition of PKC-mediated phosphorylation of P-gp.